Disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature is a central pathophysiologic event in inflammatory lung injury syndromes and results in clinically significant oxygenation derangements in critically ill patients. The goal of Project #2 is to mechanistically characterize linkage between key peripheral EC structures (dynamic cortical actin, lipid rafts, tight junctions, adherens junctions) and the cytoskeletal elements critical to the regulation of lung EC barrier function. The EC cytoskeleton is a complex array of proteins intimately involved in the cell shape changes necessary for regulation of EC barrier function. We have identified essential roles for multiple cytoskeletal linker and effector proteins in the regulation of vascular permeability through modulation of these EC cytoskeletal rearrangements: MLCK, cortactin, actinin, radixin, ZO-1. To assess the importance of these cytoskeletal elements at the periphery of the cell, Specific Aim #1 will define the structure and the regulation of dynamic cortical actin filaments rapidly polymerized at the EC periphery in response to multiple barrier enhancing stimuli. Specific Aim #2 will rigorously characterize the cytoskeletal linkage to membrane-associated lipid rafts, cholesterol-enriched microdomains within the plasma membrane that are important sites for concentrating transmembrane receptors and transducing their signals into the cell. Peripheral cell-cell contacts along the EC monolayer are essential components of barrier maintenance that serve as linkages to the actin cytoskeleton to provide both mechanical stability as well as transduction of extracellular signals into the cell. In the EC, these intercellular contacts consist primarily of two types of junctional complexestight junctions (the focus of Specific Aim #3) and adherens junctions (the focus of Specific Aim #4),whose cytoskeletal linkages will be precisely defined. This project will utilize biochemical, molecular, and cell biology approaches, murine models, translational genomic techniques, and highly novel atomic force microscopy approaches to focus intensely on characterizing the regulation of key peripheral structures by cytoskeletal linker/effector proteins. By mechanistically characterizing key membrane cytoskeletal activators, key cytoskeletal effectors, and the junctional targets affected in these processes, we expect to provide a basis for development of effective therapeutic interventions for pulmonary vascular permeability dysfunction and All.